The long term objective of this project is to define the mechanism(s) by which cardiac sarcoplasmic reticulum (SR) and sarcolemma (SL) membranes regulate sarcoplasmic Ca++. The immediate goal of this project is to determine the membrane proteins that mediate the effect of the Ca receptor protein, calmodulin, on SR and SL enzyme/channel/ion pump activities. In addition, this project will indicate the mechanisms by which protein kinase-mediated phosphorylation of SR and SL membrane proteins results in the stimulation of the Ca pump/channel activities in these membranes. This project has five specific aims. The first is to define the cardiac membrane proteins that are affinity labeled by exogenously added calmodulin and protein kinases. These results should indicate the proteins that mediate the effect of membrane protein phosphorylation on Ca channel/pump activity. The second aim is to isolate and characterize the SR and SL proteins that are affinity labeled by calmodulin. The third aim is to determine whether Ca entry blockers such as verapamil and nifedipine can modulate SL Ca channels by modifying Ca pump activity and/or the calmodulin-dependent phosphorylation of SL proteins. The fourth aim is to determine the polarization and anisotropy of fluorescence of fluorescent labeled calmodulin as it makes specific interactions with SR membranes. The effect of SR substrates and effectors (protein kinases, calmodulin) on these parameters should complement the studies in aims 1 and 2 in defining the mechanism by which calmodulin regulates SR Ca permeability. The fifth aim is to examine the effect of Ca++, calmodulin and protein kinases on the release of Ca from a heavy SR vesicle preparation that has many of the pharmacological characteristics of SR in skinned muscle fibers. Information regarding the biochemical mechanisms that regulate intracellular Ca levels in the heart may prove useful in adding to our understanding of the pathogenesis of cardiac diseases in humans, where mechanical abnormalities (cardiac failure) and disordered electrical properties (arrhythmias) are due in large measure to altered fluxes of Ca and other ions across the cardiac SL. The development of a systematic description of the mechanisms by which SR and SL transmembrane ions fluxes are regulated may aid in the formulation of improved means for clinical use in the prevention and therapy of cardiac failure and arrhythmias.